Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML.

So-called NEW ENGLISHES, distinct forms of English which have emerged in postcolonial settings and countries around the globe, have typically been regarded individually, as unique varieties shaped by idiosyncratic historical conditions and contact settings, and no coherent theory to account for these processes has been developed so far. This article argues that despite all obvious dissimilarities, a fundamentally uniform developmental process, shaped by consistent sociolinguistic and language-contact conditions, has operated in the individual instances of rerooting the English language in another territory. At the heart of this process there are characteristic stages of identity construction by the groups involved, with similar relationships between the parties in migration contact settings (i.e. the indigenous population and immigrant groups, respectively) having resulted in analogous processes of mutual accommodation and, consequently, similar sociolinguistic and structural outcomes. Outlining a basic developmental scenario, I suggest that speech communities typically undergo five consecutive phases in this processÑFOUNDATION, EXONORMATIVE STABILIZATION, NATIVIZATION, ENDONORMATIVE STABILIZATION, and DIFFERENTIATION Ñand I describe the sociolinguistic characteristics of each one. This framework is then applied to case studies of seven different countries (Fiji, Hong Kong, Malaysia, the Philippines, Singapore, Australia, and New Zealand) which, I argue, are currently positioned at different points along the developmental cycle.

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In contrast with the very well explored concept of structure-activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand complex, the structure-kinetic relationship. Here, we present a structure-kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif ("DMG" in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket.kinetic profiling | structure-based drug design T he cyclin-dependent kinase 8/cyclin C (CDK8/CycC) complex is a potent oncogene (1-4), involved in transcription and regulation of transcriptional activity (5-10), linked to epigenetic processes (11), and regarded as an attractive drug target. The recent clinical success of the small molecule inhibitors sorafenib (BAY-43006, Bayer Pharma) and imatinib (STI-571, Novartis Pharma AG) has been attributed to their deep pocket binding mode (12). The "deep pocket" (13) is adjacent to the kinase ATP binding site and accessible in protein kinases by the rearrangement of the DFG motif (a short motif composed of the residues AspPhe-Gly near the N-terminal region of the activation loop) from the active (DFG-in) to the inactive state (DFG-out) (13,14). Binding of a type II compound to such a DFG-out conformation often includes slow binding kinetics (15) with a prolonged "residence time," which is defined as the period for which a target is occupied by a compound (16). Residence time is currently considered to be a key success factor for compound optimization during drug discovery and perhaps as important as the apparent affinity such as half-inhibitory concentration (IC 50 ) or dissociation constant (K d ) (16,17). Accordingly, residence time could be a key to providing enhanced potency in vivo (18) and a means to improve the correlation of in vitro and in vivo efficacy of drugs (19). Despite the increased acceptance of the need to understand binding kinetics, the interplay between compound-target interactions and binding kinetics is in general too complex and poorly understood to enable prediction of the essential dynamic properties. The impact of the combination of kinetic data of proteininhibitor interactions with crystallographic studies has been recognized with pioneer studies such as bovine trypsin in complex with the bovine pancreatic trypsin inhibitor (20). The concept of structure-activity relationships describing the interdependency between binding affinity and compound structure has been well explored in the l...

The ongoing expansion and diversification of English, especially in the Expanding Circle, calls for a comparative analysis of these processes and a deepened theoretical understanding of this dynamism. A key question asked in this paper is whether or to what extent the 'Dynamic Model' of the evolution of Postcolonial Englishes is able to explain these processes and can be applied to Extending Circle countries as well. A decade after its first influential publication, reactions to the model are surveyed systematically, including some new applications, modifications, approaches at testing it, and some criticism. Applying the comparative-descriptive framework of this model and some of its constituent components, the progress of English is outlined in China, Korea, and (less broadly) Japan, the ASEAN, Thailand, Namibia, and Rwanda. Furthermore, it is argued that a similar dynamism is driving the widespread emergence of hybrid mixes between local languages and English and phenomena of 'poststructuralist diffusion,' English being adopted by whatever means, in fragments and unconstrained of norm concerns, driven by strongly utilitarian considerations. A tabular summary assessment compares these processes with constituents of the Dynamic Model and finds that despite some similarities it is not well suited to grasp the vibrant developments of the Expanding Circle. Instead, the notion of 'transnational attraction' is defined and proposed as an appropriate conceptual framework.

Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6-aza-benzothiophene containing compounds were developed into potent, selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252, and a key cation-π interaction with R356. Structure-based drug design resulted in tool compounds 13 and 32, which are highly potent, kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these compounds exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line. Further examination of the activity of 32 in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cause of stroke and vascular dementia. Disease-causing mutations invariably affect cysteine residues within epidermal growth factor-like repeat domains in the extracellular domain of the NOTCH3 receptor (N3(ECD)). The biochemical and histopathological hallmark of CADASIL is the accumulation of N3(ECD) at the cell surface of vascular smooth muscle cells which degenerate over the course of the disease. The molecular mechanisms leading to N3(ECD) accumulation remain unknown. Here we show that both wild-type and CADASIL-mutated N3(ECD) spontaneously form oligomers and higher order multimers in vitro and that multimerization is mediated by disulfide bonds. Using single-molecule analysis techniques ('scanning for intensely fluorescent targets'), we demonstrate that CADASIL-associated mutations significantly enhance multimerization compared with wild-type. Taken together, our results for the first time provide experimental evidence for N3 self-association and strongly argue for a neomorphic effect of CADASIL mutations in disease pathogenesis.

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an “activation helix” close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.